The project will involve a comparative study of wild type and three temperature-sensitive mutants of Neurospora crassa that have conditional defects in cytosolic ribosome biosynthesis. Each of the mutants accumulates disproportionate amounts of the two ribosomal subunits at the nonpermissive temperature. The aim of these studies is to characterize the molecular bases for the defects of the mutant strains and thus obtain information about the mechanism and regulation of ribosome production in eukaryotes. The following experiments are planned (the analytical methods for each are given in parentheses): 1) Pulse-chase studies to determine if the ribosomal subunit disproportionality results from underproduction or overproduction of a ribosomal RNA (RNA extraction, acrylamide gel electrophoresis); 2) Determination of whether the ribosomes of the mutant strains are as functional as those of the wild type (in vitro protein synthesis); 3) Determination of whether the defects in the mutant strains involve temperature-sensitive synthesis or function (RNA isolation, acrylamide gel electrophoresis); 4) Examination of the kinetics of rRNA synthesis in the mutants growing at their permissive and non-permissive temperatures to see how this process differs qualitatively and quantitatively from that in the wild type (nuclei isolation, RNA extraction, acrylamide gel electrophoresis); 5) Studies to see if the ribosome defects in the mutants result from altered ribosomal proteins, altered sequence of addition of the ribosomal proteins to the nascent RNA, altered methylation of the rRNAs, altered activity of S-adenosyl methionine synthetase or altered activity of rRNA methylases. (Two-dimensional acrylamide gel electrophoresis, ribosomal protein extraction, chromatin isolation, DEAE-cellulose column chromatography and RNA fingerprint analysis). In addition the cold-sensitive, ribosome biosynthesis mutant, crib-1, will be studied to determine whether there is coordinate regulation of the synthesis of 17S rRNA and the ribosomal proteins of the 37S subunit that is independent of the regulation of synthesis of the rRNAs and ribosomal proteins of the 60S subunits.